The sterol response element binding protein (SREBP) transcription factors have been identified as key regulators of genes involved in both cholesterol and lipid homeostasis. Because of the prominence of aberrant cholesterol and lipid homeostasis in the metabolic dysregulation underpinning cardiovascular disease and diabetes, transcriptional regulators of cholesterol/lipids such as SREBPs could potentially represent important therapeutic targets to ameliorate these diseases. Interestingly, small regulatory RNAs termed microRNAs (miRNAs) have recently also been found to represent important regulators of eukaryotic gene expression. One miRNA, miR-122, was shown in recent studies to modulate cholesterol levels in mice and non-human primates. These studies have highlighted the critical role played by miRNAs in mammalian physiology with relevance to human health. Intriguingly, we have obtained preliminary data suggesting that miR-33a/b, miRNAs that are embedded within intronic sequences in both SREBP-1 and -2 genes in humans, regulate cholesterol homeostasis in mammals in coordination with the SREBP host gene products. Our results show that miR-33 miRNAs target the ABCA1 cholesterol transporter for translational repression, resulting in lowered ABCA1 protein levels and decreased cholesterol efflux. As SREBPs promote cholesterol uptake and synthesis through the transactivation of the LDL receptor gene and cholesterol biosynthesis genes, inhibition of cholesterol efflux by miR-33-dependent down-regulation of ABCA1 acts in cooperation with SREBPs to effectively boost intracellular cholesterol levels. We propose a series of in vitro and in vivo studies to further determine the functional role of miR-33 in controlling cholesterol and lipid homeostasis in cooperation with SREBPs. These studies could have important ramifications for our understanding of the integration and coordination of miRNA and transcription factor gene regulation and will facilitate novel therapeutic strategies to increase HDL synthesis and reverse cholesterol transport in patients with cardiovascular disease. The Specific Aims are: 1. To examine the co-expression of miR-33 and its host gene SREBP in cells and tissues. 2. To determine the role of miR-33 in regulation of cholesterol trafficking. PUBLIC HEALTH RELEVANCE: Cholesterol and fatty acids play critical functional roles in many physiological processes in metazoans, such as modulating membrane fluidity, serving as signaling molecules, and providing energy storage in the form of triglycerides. Abnormal cholesterol and lipid levels have been linked to increasingly prevalent diseases, including atherosclerosis, obesity, type II diabetes, and hypertension (all associated with metabolic syndrome), as well as lipodystrophy, Alzheimers Disease, and certain cancers, underscoring the importance of understanding fully how cholesterol and lipid homeostasis are regulated and maintained. We and others have investigated cholesterol and lipids are controlled by gene switches termed sterol regulatory element binding protein (SREBP). Intriguingly, we have noted the presence of microRNAs (miR- 33a/b) within intronic sequences in the human SREBP-2 and -1 genes, respectively. Preliminary studies yielded the surprising finding that miR-33a/b appear to repress the cholesterol transporter ABCA1, a key regulator of HDL synthesis that is involved in preventing atherosclerosis. As SREBPs promote cholesterol uptake and synthesis, we predict that miR-33-mediated inhibition of ABCA1 and cholesterol efflux acts in cooperation with SREBPs to boost intracellular cholesterol levels. We propose a series of in vitro and in vivo studies that will delineate the role of miR-33 in controlling cholesterol/lipid homeostasis in cooperation with SREBPs. These studies should provide novel therapeutic strategies aimed at increasing HDL synthesis and cholesterol clearance in patients with cardiovascular disease.